In these studies we define a new type of IL-10-producing regulatory T cell that is induced by Dectin receptor-stimulated dendritic cells. These cells differ from previously described IL-10-producing regulatory cells both with respect to their mode of induction and the molecular events that underlie their activation of IL-10 gene transcription. On this basis we have termed them Tr2 regulatory T cells. With respect to their mode of induction we showed that Tr2 cells are induced by zymogen-depleted yeast extracts and by the hyphal form of C. albicans, both of which express 1,3-beta glucan, the ligand of Dectin-1. With respect to IL-10 gene transcription, Tr2 cells undergo two interlocking molecular processes that together result in high level IL-10 production. The first involves T cell production of IL-4 and activation of Th2 genes including STAT6 and GATA3. On this basis, T cells that cannot produce IL-4, or express STAT6 and GATA3 cannot be induced to become high level IL-10 producers following Dectin-1 stimulation of inducing DCs. The key element in this signaling pathway is most likely GATA3 since we show that GATA3 binds to the Tr2 cell IL-10 promoter at two sites, i.e., at a distal site where it acts as a direct transcription factor and at the proximal site where it acts indirectly on transcription as a epigenetic factor that augments histone acetylation. The second and more unique process guiding IL-10 transcription in Tr2 cells was revealed in studies showing that the TORC1 arm of the mTOR signaling pathway is a critical component of IL-10 production in such cells. This was shown quite definitively by the fact that IL-10 production in T cells stimulated by Dectin-1-activated DCs is subject to dose-dependent inhibition by the presence of rapamycin. The mechanism of this inhibitory effect was found to involve another quite distinctive aspect of IL-10 transcription in Tr2 cells, namely that such transcription depends on expression of a particular C/EBP isoform whose induction depends on TORC1 signaling. The series of studies that led to these conclusions began with micro-array analyses in which we examined gene expression in Tr2 cells as well as in Tr1 and Th2 cells. These studies showed that gene expression in Tr2 cells was distinct from that in Tr1 and Th2 cells and that IPA analysis of such expression showed that C/EBP signaling was among the several signaling pathways that could underlie this distinct expression pattern. In subsequent studies to examine this possibility we showed first that stimulation of T cells from mice with targeted deletion of C/EBP stimulated under Tr2 conditions led to greatly decreased IL-10 production as compared to similarly stimulated WT cells. In addition, we showed that T cells from C/EBP-deficient mice stimulated under Tr2 conditions in which C/EBP levels were partially repleted by culture with a retrovirus expressing isoforms of C/EBP led to recovery of IL-10 production if the repleting retrovirus expressed the LIP isoform of C/EBP but not if the virus expressed the LAP isoform of C/EBP. These studies thus provided strong evidence that the LIP, but not the LAP isoform of C/EBP is the isoform of C/EBP that is a necessary component of IL-10 transcription in Tr2 cells. Finally, we could relate the relation of TORC1 signaling to IL-10 production in Tr2 cells with studies that showed that TORC1 signaling led to the phosphorylation of eukaryote initiation factor ((elf)-4E), a factor that has been shown to regulate C/EBP translation into LAP and LIP and is necessary for the LIP expression. Thus, in the absence of TORC-1 signaling due to the presence of rapamycin, LIP translation from C/EBP is virtually absent and, as a result, IL-10 production in Tr2 cells is greatly inhibited. In parallel studies, we investigated the mechanism of how LIP regulates IL-10 production in Tr2 cells. These initially centered around studies with an IL-10 promoter-luciferase construct already alluded to above and showed that promoter activity was maximally stimulated by the presence of plasmids expressing CREB1 and LIP and in fact deletion of binding sites for these factors led to greatly reduced promoter activity. Since the CREB1 and LIP binding sites in the promoter are adjacent to one another and CREB1 had been shown previously to bind to C/EBP we reasoned that the LIP1/CREB1 cooperativity was due to facilitated binding of one or both factors to the IL-10 promoter. This hypothesis was subsequently supported by EMSA studies that showed that CREB1-LIP protein complexes extracted from the nucleus of HEK293 cells (pre-transfected with CREB1 and LIP expressing plasmids) bound to the DNA sequence found in the IL-10 promoter binding these transcription factors under physiologic conditions; in contrast, a similarly obtained CREB1-LAP complex had a poor capacity to bind to this sequence. These findings were accompanied by studies showing that C/EBP and CREB1 binding to the IL-10 promoter in Tr2 cells as determined by CHiP studies was enhanced in cells expressing LIP and LAP as compared to cells expressing only LAP, indicating the CREB1 binding is enhanced by complex formation with LIP. These studies support the conclusion that TORC1 signaling in nascent Tr2 cells leads to high IL-10 production because such signaling generates LIP-CREB1 complexes and augmented binding of these transcription factors to the IL-10 promoter. In summary, these studies establish that Dectin-1 stimulation of DCs elicits an new type of regulatory T cell that this is exquisitively dependent on mTor (TORC1) signaling. In related studies of Tr2 cell generation we showed that a COX2 inhibitor, Celecoxib, greatly enhances induction of Tr2 cells by inducing mTor activation of 4-EBP-1 and the LIP form of C/EBP-beta. We then showed that Celecoxib acts in this context as a PGE2 inhibitor that signals via the EP2 receptor to inhibit mTor and activation of 4EBP1/generation of LIP. Then in separate studies we showed that PGE inhibition is mediated by PKA induction and that PKA inhibition mimics the effect of Celecoxib. Thus, the Celecoxib is acting as an inhibitor of an inhibitor of IL-10 production (PGE2). We next turned our attention to defining the clinical important of Tr2 induction in the context of C. albicans infection . In this context we showed that C. albicans renal infection is associated with IL-10 producing CD4 T cells in the renal tissue and mice that have CD4 T cells that cannot produce IL-10 (CD4Cre/ IL:-10 flox mice)i.e., mice that cannot produce Tr2 cells, exhibit better survival of infection than wild type mice. Conversely, treatment of mice with Celecoxib, an agent that augments Tr2 cell generation, have reduced survival. Thus, generation of Tr2 cells limits the pro-inflammatory (protective) effect of C. albicans infection. Lastly, we have been conducting on-going studies to define how Dectin-1 stimulated dendritic cells signal T cells to initiate a transcriptional program that results in high level IL-10 production, Based on the fact that T cells undergo both Th2 and mTor signaling to generate the transcription factors mediating IL-10 production in Tr2 cells (GATA-3 and C/EBP-beta) we most recently conducted studies to determine if TSLP might be the dendritic cell signal inducing Tr2 cells. However, this proved not to be the case. Currently, we are setting up RNAseq studies to determine the factors produced by Dectin-1 stimulated cells when the latter are co-stimulated by interacting T cells (e.g. p40 ligand and other T cell co-stimulators) and then to test likely Tr2-inducing factors so revealed.